The hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus of the Flaviviridae family that causes more deaths in the US than HIV. Infection with HCV is the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and currently affects ~180 million people worldwide, ~4 million of whom reside in the US. No protective vaccines exist against HCV. The current standard of care is a combination of pegylated interferon and ribavirin, with limited effectiveness and severe side effects. The recently FDA- approved protease inhibitors Telaprevir and Boceprevir have to be administered with this standard treatment to achieve sustained viral clearance so that even the newest therapy is still associated with serious side effects. The recent discovery by our collaborator Peter Sarnow (Stanford University) of a remarkable protective role for microRNA (miRNA) miR-122 in HCV replication has paved the way for anti-miRs, which inhibit endogenous miRNAs, as a novel class of HCV drugs. In addition, our collaborator Brian Johnston from SomaGenics Inc. recently developed several small interfering RNAs (siRNAs) as well as short small hairpin RNAs (sshRNAs) that mediate efficient RNA interference (RNAi) against HCV in cell culture. The primary objective of this R21 proposal is to dissect the underlying intracellular mechanisms of these promising small-RNA anti-HCV drugs, with the long-term goal of further improving their efficacy. To this end, we will bring to bear the powerful capabilities of Single molecule, High-Resolution Localization and Counting (iSHiRLoC), an innovative probe concept we recently developed for detecting the trafficking and assembly of single miRNA molecules inside live cultured cells. iSHiRLoC entails single particle tracking of physiologically relevant lw numbers of microinjected, inconspicuously fluorophore-labeled RNAs to record their spatiotemporal distribution and count their stoichiometry upon RNA:protein (RNP) complex assembly. We propose to expand iSHiRLoC to two colors and study HCV biology and inhibition at an unprecedented single molecule level, by pursuing the following three Specific Aims: (i) We will probe the mechanism of miR-122-mediated intracellular protection of HCV RNA by using two-color iSHiRLoC in cultured, miR-122-free HepG2 hepatocytes and comparing the spatiotemporal distributions and assembly stoichiometries of Cy5-labeled miR-122, microinjected in conjunction with either Cy3-labeled subgenomic HCV RNA or Cy3-labeled AldoA target mRNA. (ii) We will probe the mechanism of siRNA- and sshRNA-mediated intracellular RNA interference against HCV by applying two-color iSHiRLoC to siRNAs and sshRNAs already validated against HCV in hepatocytes by SomaGenics Inc. (iii) We will observe the tug-of-war between miR-122-mediated protection of and RNAi against HCV RNA by performing a precise intracellular titration of Cy3-labeled HCV RNA with varying amounts of unlabeled miR-122 in the presence of our most effective Cy5-labeled siRNA/shRNA to directly observe the evolution of the HCV RNA spatiotemporal distribution as the tug-of-war between the two small RNAs plays out.